Comparative proteomic profiling of human osteoblast‐derived extracellular matrices identifies proteins involved in mesenchymal stromal cell osteogenic differentiation and mineralization

Abstract

The extracellular matrix (ECM) is a dynamic component of tissue architecture that physically supports cells and actively influences their behavior. In the context of bone regeneration, cellsecreted ECMs have become of interest as they reproduce tissue-architecture and modulate the promising properties of mesenchymal stem cells (MSCs). We have previously created an in vitro model of human osteoblast-derived devitalized ECM that was osteopromotive for MSCs. The aim of this study was to identify ECM regulatory proteins able to modulate MSC differentiation to broaden the spectrum of MSC clinical applications. To this end, we created two additional models of devitalized ECMs with different mineralization phenotypes. Our results showed that the ECM derived from osteoblast-differentiated MSCs had increased osteogenic potential compared to ECM derived from undifferentiated MSCs and non-ECM cultures. Proteomic analysis revealed that structural ECM proteins and ribosomal proteins were upregulated in the ECM from undifferentiated MSCs. A similar response profile was obtained by treating osteoblast-differentiating MSCs with Activin-A. Extracellular proteins were upregulated in Activin-A ECM, whereas mitochondrial and membrane proteins were downregulated. In summary, this study illustrates that the composition of different MSC-secreted ECMs is important to regulate the osteogenic differentiation of MSCs. These models of devitalized ECMs could be used to modulate MSC properties to regulate bone quality. K E Y W O R D SActivin-A, bone regeneration, extracellular matrix, mesenchymal stromal cells Due to the role of the ECM on actively modulating MSC osteogenic differentiation and to the increasing interest of using MSCs for cell-based therapies, the aim of our study was to identify ECM proteins that are involved in osteoblast differentiation and mineralization making these proteins suitable candidates to control bone quality. To this end, we created in vitro ECM models with extremely different mineralization phenotypes, in order to get more insights into the effect of the ECM composition on MSC behavior.We have previously created an in vitro model of human MSC- 2 | MATERIALS AND METHODS | Cell culture and ECM preparationsHuman bone marrow-derived MSCs were used to produce the devitalized ECM as previously described (Baroncelli M., unpublished data). Briefly, commercially available MSCs (PT-2501, Lonza, Walkersville, MD) from a single donor at passage 7, were cultured in growth media (alpha-Mem phenol-red free (GIBCO, Paisley, UK), 10% fetal bovine serum) for 2 days. Then, MSCs were cultured for 11 days by supplementing the growth medium with 100 nM dexamethasone and 10 mM β-glycerophosphate (Sigma, St. Louis, MO) to induce the osteogenic differentiation. MSCs were devitalized before the onset of mineralization by freeze-thaw cycles and DNAse treatment (10 U/ml; Sigma-Aldrich), followed by extensive washings with Phosphate Buffer Saline (PBS, Gibco BRL, Carlsbad, CA) and sterile air drying, before freezing and subsequent use.In ...

Systems biology analysis of osteogenic differentiation behavior by canine mesenchymal stem cells derived from bone marrow and dental pulp

“…Interestingly, most of the annotated proteins in 3 categories showed different expression levels between 2 cell types, and the expression patterns were very dynamic during the osteogenic induction process which suggested the distinct underlying mechanisms and potential functional proteins/components for controlling their osteogenicity. These findings were correlated with the osteogenic behavior of several MSCs derived from different sources that uniquely contained their osteogenicity and underlying differentiation processes/pathways 34,[38][39][40] .…”

“…Previous studies mostly focused on individual osteogenic differentiation potential of the cells, rather than comparing and clarifying their osteogenic differentiation behavior 8,9,31,32 . To address this concern, comparative proteomics-based systems biology analysis was performed for dissecting the osteogenic differentiation behavior of cBM-MSCs and cDPSCs in vitro, since quantitative proteomics analysis has been proposed as a promising tool for comprehensively analyzing an osteogenic differentiation by many MSC-based osteogenic models 33,34 . Time-series analysis (day 0 vs. day 7 vs. day 14) of osteogenic differentiation behavior by cBM-MSCs and cDPSCs was conducted to thoroughly clarify this issue.…”

Human mesenchymal stromal cells in adhesion to cell‐derived extracellular matrix and titanium: Comparative kinome profile analysis

“…Human bone marrow‐derived MSCs were used to prepare the osteopromotive devitalized ECM as previously described (Baroncelli et al, ). Briefly, MSCs (5,128 viable cells/cm 2 ; PT‐2501; Lonza, Walkersville, MD) from a single donor at passage seven were cultured in growth medium for 2 days (α‐Mem phenol‐red free [Gibco, Paisley, UK], 10% foetal bovine serum), and osteogenically differentiated for 11 days (culture medium supplemented with 100 nM of dexamethasone and 10 mM of β glycerophosphate [Sigma‐Aldrich, St. Louis, MO] to induce the deposition of the ECM.…”

Engineering Stem Cell-Derived Extracellular Matrices: Decellularization, Characterization, and Biological Function

“…106 These data indicate that tsECMs can at least partially recapitulate the signaling network of the tissue source to support stem cell self-renewal or direct lineage-specific differentiation. 29,107 Signaling Mediated by scECMs Different from tsECMs, scECMs contain unique signaling networks that regulate self-renewal and lineage specification during tissue development and recapitulate specific stem cell microenvironment. 12 Understanding these signaling networks should better control the in vitro culture environment and coax the in vivo development of the transplanted stem cells.…”

“…and immobilized molecules (e.g., TGF-b, CTGF, CYR61) are responsible for the osteoinductive properties of MSC-ECMs. 107 The chondrogenic potential of MSCs can be enhanced on MSC-derived ECMs possibly due to the upregulation of TGF-b receptor II. 121 MSC-ECMs have also been found to stimulate cytokine secretion, including angiopoietin-1 (ANG-1), SDF-1, and IL-8 from the reseeded MSCs, and ANG-2, VEGF, and Heng et al 141 AC, ascorbic acid; A-MSC, adipose tissue-derived mesenchymal stem cells; ANG-1, angiopoietin 1; BM-MSC, bone marrow mesenchymal stem cells; DFSCs, dental follicle stem cells; DPP, dentine phosphophoryn; DSP, dentin sialophosphoprotein; IBSP, integrin-binding sialoprotein; IL-8, interleukin 8; K14, keratin 14; LAMB1, laminin subunit beta 1; NPC, neural progenitor cell; PEDF, pigment epithelium-derived factor; phospho-TGF-bRII, phosphorylated form of transforming growth factor beta receptor II; RPL13, ribosomal protein L13; SDF-1, stromal cell-derived factor 1.…”